Patients and procedures
Eligible patients were required to have pathologically-confirmed NSCLC and sufficient tissue for analysis. ALK-positivity was assessed with either VENTANA IHC or RT-PCR. Clinical and pathologic data prospectively collected for analyses included age at the time of diagnosis, gender, smoking status, stage, histology, specimen type, and EGFR status according to the new International Association for the Study of Lung Cancer, American Thoracic Society, and European Respiratory Society multidisciplinary classification. A subset of patients received crizotinib treatment (250 mg twice daily) and had clinical data available on the outcome. Imaging data were independently reviewed by authors to evaluate the treatment responses according to the Response Evaluation Criteria in Solid Tumors (RECIST; version 1.1). Progression-free survival (PFS) was calculated from the date of initiating targeted drug treatment to radiologic or clinical observation of disease progression. This study was approved by the Ethics Committee of Fujian Cancer Hospital (Fujian Medical University Cancer Hospital, Fuzhou Fujian, China) and written informed consent was obtained from each participant before the initiation of any study-related procedure.
VENTANA immunohistochemistry
Immunohistochemistry was carried out on a fully automated VENTANA Benchmark XT stainer (VENTANA Medical Systems; Roche Group, Tucson, AZ, USA) using the pre-diluted VENTANA anti-ALK (D5F3) rabbit monoclonal primary antibody, together with the Optiview DAB IHC detection and Optiview amplification kits (VENTANA Medical Systems; Roche Group, Tucson, AZ, USA). Each case was also stained with a matched rabbit monoclonal negative control immunoglobulin antibody. A binary scoring system was adopted for evaluating the staining results. The presence of strong granular cytoplasmic staining in tumor cells (any percentage of positive tumor cells) was deemed to be ALK-positive, while absence of strong granular cytoplasmic staining in tumor cells was deemed to be ALK-negative.
RNA preparation
The RNA was extracted from lung tumors per standard protocols (RNeasy Mini Kit; Qiagen, Hilden, Germany or AmoyDx RNA Kit; Amoy Diagnostics Co., Xiamen, China). This extraction method was optimized by the manufacturer to reverse formaldehyde modification without further RNA degradation and has been shown to be an efficient method to obtain RNA of sufficient quantity for PCR amplification in our laboratory.
ALK rearrangement detection
RT-PCR was used to detect the ALK rearrangement. The ALK rearrangement mRNA was detected using the AmoyDx EML4-ALK Fusion Gene Diagnostic Kit (Cat No. ADx-FF04), which is designed to detect 21 types of known ALK rearrangements, including E6;A19, E6;A20, E6ins33;A20, E6;ins18A20, E13;A20, E13;ins69A20, E20;A20, E20;ins18A20, E14ins11;del49A20, E14;del14A20, E14;del38A20, E15del60;del71A20, E2;A20, E2;ins117A20, E3;ins53A20, E17;ins30A20, E17ins61;ins34A20, E17ins65;A20, E17;ins68A20, E17del58;ins39A20, and E18;A20. In brief, the mRNA extracted from the previous step was reverse-transcribed to cDNA at 42 °C, followed by PCR amplification. The PCR condition of the cDNA was as follows: initial denaturation at 95 °C for 5 min, followed by 95 °C for 25 s, 64 °C for 20 s, 72 °C for 20 s to ensure the specificity; and up to 31 cycles at 93 °C for 25 s, 60 °C for 35 s, and 72 °C for 20 s. The details are described in our previous studies [16, 17].
Targeted next-generation sequencing
For 17 patients in whom the two methods were inconsistent, targeted region capture combined with NGS was performed. Genomic DNA sequencing libraries were prepared using the protocols recommended by the Illumina TruSeq DNA Library Preparation Kit (Illumina, San Diego, CA, USA). For samples close to the minimum input requirement, additional pre-capture PCR cycles were performed to generate sufficient PCR product for hybridization. The libraries were hybridized to custom-designed probes (Integrated DNA Technology, Coralville, IA, USA), including all exons of 170 genes and selected introns of ALK, RET, and ROS1 for the detection of genomic rearrangements. DNA sequencing was performed on a HiSeq3000 sequencing system (Illumina, San Diego, CA, USA) with 2 × 75 bp paired-end reads. The reads were aligned to the human genome build GRCh37 using a Burrows–Wheeler aligner (BWA). Somatic single nucleotide variant (sSNV) and indel calls were generated using MuTect and GATK, respectively. Somatic copy number alterations were identified with CONTRA. Genomic rearrangements were identified by the software developed in-house analyzing chimeric read pairs.
Statistical analysis
A Chi square or Fisher’s exact test was used to analyze correlations between ALK status and the clinicopathologic factors. The response rate among subgroups and survival were described with Kaplan–Meier methodology and the log-rank test was used to compare survival among subgroups. Statistical analysis was performed using SPSS (version 19.0 software; IBM, Armonk, NY, USA). All p values were two-sided, and a p < 0.05 was considered statistically significant.
